The invention relates to methods for the identification of compounds that modulate the activity of bimC and use of such methods for the identification of therapeutic agents and agricultural compounds.
There is a compelling need to prevent and treat opportunistic fungal infections that result in a variety of manifestations, many of which are fatal if untreated. Indeed, the 1980s witnessed an epidemic rise in Candida infections. A steep rise in the incidence of Aspergillus infections has occurred during the 1990""s. Similar rises in zygomycosis, cryptococcosis, histoplasmosis and fusaria infection have also been noted. The reasons for the rise in fungal infections are several, but a key factor is the growing population of immuno-compromised individuals. This group includes patients with HIV disease (AIDS), older patients, patients who have undergone invasive surgery, transplant patients and burn victims.
Accordingly, the need for novel antifungal drugs is clear. At present there are two accepted therapies, namely, amphotericin B and the azole family of compounds. Both are effective for certain fungal infections but each have drawbacks-amphotericin B is nephrotoxic and there are increasing numbers of azole resistant strains. These drugs act by either directly or indirectly disrupting the fungal cell wall (or its underlying membrane), which has been the target of choice for antifungal agents.
As the population of immunosuppressed individuals increases, so do the numbers and types of fungal infections noted in these patients. Although candidiasis remains the most common fungal infection in immunosuppressed patients, aspergillosis, zygomycosis, and other filamentous fungal infections are a major problem for an increasing number of patients. The endemic mycoses, especially histoplasmosis and coccidioidomycosis, also constitute a risk for patients. Those at particular risk for such infections are those with AIDS, those having undergone bone marrow or organ transplants, those receiving chemotherapy and others who have had debilitating illness, severe injury, prolonged hospitalization, or long-term treatment with antibacterial drugs.
According to the CDC""s National Nosocomial Surveillance System, the rate of hospital-related fungal infections nearly doubled between 1980 and 1990. In 1997, an estimated 240,000 individuals showed clinical symptoms of endemic mycoses. With the current approaches to treatment (primarily amphotericin B and the azoles) the mortality rate in patients with systemic fungal infections ranges from 30-100%, depending on the infection.
The severity of the fungal infections increases as the immune system becomes more dysfunctional. Fungi are among the most ubiquitous pathogens seen in patients with AIDS; virtually all major fungal pathogens cause disease in HIV-positive patients. The majority of untreated HIV-positive patients experience at least one episode of fungal infection and many fungal infections are AIDS-defining illnesses in AIDS patients.
Many emerging fungal pathogens are resistant to the currently available antifungal agents and, thus, pose a special risk for immunocompromised patients. Furthermore, as existing therapies are limited in the range of fungal pathogens against which they show efficacy, and as identification of the fungal pathogen infecting an individual is slow relative to the urgent need to control the infection, there is a critical need to develop broad-spectrum antifungal therapeutics.
Clinically relevant mycotic diseases include candidiasis, aspergillosis, cryptococcosis, endemic mycoses and infections caused by a host of new, emerging fungal pathogens. The standard of care for the more common mycoses employs amphotericin B and the triazole family of therapeutics. These treatments are far from ideal; substantial side effects are seen with amphotericin B. For the azoles problematic issues include increasingly resistant strains and limited bioavailability. Finally, for some of the endemic mycoses as well as new emerging fungal pathogens, no appropriate therapeutics are available. The best prospect for combating existing and emerging threats is through the development of broad-spectrum antifungals.
Given the increased numbers of patients manifesting mycotic diseases, combined with the lack of effective antifungals and the increase in azole resistant isolates, there is a compelling need for novel antifungals. The present invention provides a novel method to identify such antifungals.
The present invention provides methods to identify candidate agents that bind to a target protein or act as a modulator of the binding characteristics or biological activity of a target protein. In one embodiment, the method is performed in plurality simultaneously. For example, the method can be performed at the same time on multiple assay mixtures in a multi-well screening plate. Furthermore, in a preferred embodiment, fluorescence or absorbance readouts are utilized to determine activity. Thus, in one aspect, the invention provides a high throughput screening system for detecting modulators of bimC enzyme activity.
Preferably, the target protein either directly or indirectly produces ADP or phosphate. More preferably, the target protein comprises bimC or a fragment thereof.
The method further comprises adding a candidate agent to a mixture comprising the target protein under conditions that normally allow the production of ADP or phosphate. The method further comprises subjecting the mixture to an enzymatic reaction that uses said ADP or phosphate as a substrate under conditions that normally allow the ADP or phosphate to be utilized and determining the level of activity of the enzymatic reaction as a measure of the concentration of ADP or phosphate. The phrase xe2x80x9cuse ADP or phosphatexe2x80x9d means that the ADP or phosphate are directly acted upon by detection reagents. In one case, the ADP, for example, can be hydrolyzed or can be phosphorylated. As another example, the phosphate can be added to another compound. As used herein, in each of these cases, ADP or phosphate is acting as a substrate. A change in the level between the presence and absence of the candidate agent indicates a modulator of the target protein.
Also provided are modulators of the bimC subfamily of kinesins including antifungal agents. The agents and compositions provided herein can be used in variety of applications which include the formulation of sprays, powders, and other compositions in the formulation of antifungal preparations which can be used to prevent the growth of fungi. Also provided herein are methods of preventing and treating fungal infections.